title: Immunolocalization of Hsp60 in Legionella pneumophila.
abstract: One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including L. pneumophila, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L. pneumophila grown in vitro, or in vivo inside of HeLa cells. For comparative purposes Escherichia coli, expressing recombinant L. pneumophila Hsp60, was employed. In contrast to E. coli, where Hsp60 was localized exclusively in the cytoplasm, in L. pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked L. pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by L. pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in L. pneumophila and further suggest both the existence of a novel secretion mechanism (not present in E. coli) and a potential role in pathogenesis.
authors.affiliation: Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: R A
authors.initials: RA
authors.lastname: Garduño
authors.forename: G
authors.initials: G
authors.lastname: Faulkner
authors.forename: M A
authors.initials: MA
authors.lastname: Trevors
authors.forename: N
authors.initials: N
authors.lastname: Vats
authors.forename: P S
authors.initials: PS
authors.lastname: Hoffman
mesh_terms: D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001886:Bordetella pertussis / Q000737:chemistry / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry; D018834:Chaperonin 60 / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D016952:Legionella pneumophila / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D018834:Chaperonin 60; D011993:Recombinant Fusion Proteins